Process for the preparation of cephalosporin

ABSTRACT

A process comprising the treatment of a compound selected from the group consisting of   WHERE R represents alkyl having up to seven carbon atoms, phenyl, X-substituted phenyl, naphthyl, X-substituted naphthyl, pyridyl, pyrryl, furyl, thienyl, pyridyl-lower alkyl, pyrryllower alkyl, furyl-lower alkyl, thienyl-lower alkyl,   D R A W I N G WHEREIN R2 represents alkyl having up to seven carbon atoms, phenyl, X-substituted phenyl, naphthyl, cyclohexadienyl (e.g., 1,4-cyclohexadienyl, 1,3-cyclohexadienyl, and 2,4cyclohexadienyl), X-substituted naphthyl, pyridyl, pyrryl, furyl, thienyl, pyridyl-lower alkyl, pyrryl-lower alkyl, furyl-lower alkyl, or thienyl-lower alkyl; R3 represents lower alkyl, monocyclic aryl, or monocyclic aryl-lower alkyl; R4 and R5 each represent hydrogen, lower alkyl, monocyclic aryl, or monocyclic aryl-lower alkyl; n is 1, 2, or 3; and X represents lower alkyl, lower alkoxy, or halo; and R1 represents triphenylmethyl, diphenylmethyl, or benzyl, said treatment comprising heating the above compounds to a temperature of about 25* C. to about 100* C. at a pH of about 6 to 11.5. Products prepared by means of the above reaction possess antibacterial activity against a large number of micro-organisms and are intermediates for the synthesis of related compounds (such as cephalothin, cephalexin, cephaloridine) which possess utility known to the art.

United States Patent Saul Lewis Neidleman Lawrence Township;

Jerold Alan Last, Princeton; Samuel Cheng Pan, Metuchen; Joseph EdwardDoltini, N. Brunswick, all of NJ.

Jan. 17, 1969 Dec. 14, 1971 E.R. Squibb & Sons, Inc.

New York, NY.

[72] inventors [21 Appl. No. [22] Filed [45] Patented [73] Assignee [54]PROCESS FOR THE PREPARATION OF Primary ExaminerNicholas S. RizzoAttorneysLawrence S. Levinson, Merle J. Smith and Theodore J. CriaresABSTRACT: A process comprising the treatment of a compound selected fromthe group consisting of;

and

wherein R represents alkyl having up to seven carbon atoms, phenyl,X-substituted phenyl. naphthyl, X-subslituted naphthyl, pyridyl, pyrryl,furyl, thienyl, pyridyl-lower alkyl, pyrrl-lower alkyl, furyl-loweralkyl, thienyl-lower alkyl,

wherein R represents alkyl having up to seven carbon atoms, phenyl,X-substituted phenyl, naphthyl, cyclohexadienyl (e.g.,1,4-cyclohexadienyl, 1,3-cyclohexadienyl, and 2,4- cyclohexadienyl),X-substituted naphthyl, pyridyl, pyrryl, furyl, thienyl, pyridyl-loweralkyl, pyrryl-lower alkyl, furyl-lower alkyl, or thienyl-lower alkyl; Rrepresents lower alkyl, monocyclic aryl, or monocyclic aryl-lower alkyl;R and R each represent hydrogen, lower alkyl, monocyclic aryl, ormonocyclic aryl-lower alkyl; n is l, 2, or 3; and X represents loweralkyl, lower alkoxy, or halo; and R represents triphenyl- I methyl,diphenylmethyl, or benzyl, said treatment comprising heating the abovecompounds to a temperature of about 25 C. to about 100C. at a pH ofabout 6 to 11.5. Products prepared by means of the above reactionpossess antibacterial activity against a large number of micro-organismsand are intermediates for the synthesis of related compounds (such ascephalothin, cephalexin, cephaloridine) which possess utility known tothe art.

PROCESS FOR THE PREPARATION OF CEPHALOSPORIN This invention relates to anovel process for the preparation of3-deacetyl-N-substituted-7-aminocephalosporanic acids which haveactivity as antimicrobial agents.

One of the synthetic preparations of cephalosporins requires that anintermediate having the formula A be synthes- Up to the present nosatisfactory method has been available to open the lactone ring of suchcompounds, since simultaneous destruction of the B-lactam ring occurs[cf. Topics in Pharmaceutical Sciences, 1, 33-51, lntersciencePublishers (l968)]. In the absence of a suitable process for opening thelactone ring, therefore, efforts at the development of a total synthesisof cephalospon'ns by this relatively simple route have beenuneconomical.

It has now been discovered that the lactone ring may be opened insatisfactory yield by the process of the present invention.

It is, accordingly, an object of the present invention to preparecompounds having the formula wherein R represents alkyl having up toseven carbon atoms, phenyl, X-substituted phenyl, naphthyl,X-substituted naphthyl, pyridyl, pyrryl, furyl, thienyl, pyridyl-loweralkyl, pyrryl-lower alkyl, furyl-lower alkyl-thienyl-lower alkyl,

NH; NH

E1 7 Li.

All four halogens are contemplated. The term lower alkyl" as employedherein includes both straight and branched chain radicals of less thaneight carbon atoms. Lower alkyl groups (All are exemplified by methyl,ethyl, propyl, isopropyl, n-butyl, l l -dimethylbutyl and n-hexyl.

By monocyclic aryl is meant phenyl and substituted phenyl radicals suchas lower alkyl phenyl, as exemplified by o-, m-, or p-tolyl andethylphenyl, di(lower alkyl)-phenyl as exemplified by p-xylyl, loweralkoxyphenyl as exemplified by methoxyphenyl and propoxyphenyl andhalophenyl as exemplified by chlorophenyl, bromophenyl, iodophenyl andfluorophenyl.

Further contemplated within this invention are the preparation of saltsof the above acids with various pharmaceutically acceptable cations asis well known in the art.

As pharmaceutically acceptable cations may be mentioned metallic cationssuch as sodium, potassium, calcium and aluminum and organic aminecations such as trialkylamines, e.g., triethylamine, procaine,dibenzylamine, N-benzyl-B- phenethylamine, l -ephenamine, N,Ndibenzylethylenediamine, N-(lower)alkylpiperidines, e.g., N-ethylpiperidine, quaternary ammonium ions (e.g., tetramethylammonium,tetraethylammonium, pyridinium, and the like), and other amines whichhave been used to form salts with benzylpenicillin.

Compounds prepared by means of the process of this invention (i.e., thecompounds of Formulas land II) are physiologically active substanceswhich have a high degree of antibacterial activity against a largenumber of micro-organisms, including Gram positive and Gram negativemicro-organisms such as Staphylococcus aureus, Escherichia coli,Klebsiella pneumoniae, Aerobacter aerogenes, and Shigella sonnei.

For these purposes, they may be administered orally or parenterally insuch form as tablets, capsules, injectables, or the like, byincorporating the appropriate dosage of the compound with carriersaccording to standard pharmaceutical practice. Dosages for variousmammillary species (e.g., rats, dogs, cats, cattle, horses, and soforth) would be from about 0.01 to about 1.5 g./kg. daily, administeredonce to several times a day.

In addition, the compounds of this invention are useful as supplementsto animal feeds, such as for poultry, cattle, and swine (particularlyweanling pigs) as an aid in increasing growth rate, improved feedefficiency and in the suppression of infections during periods ofstress, such as weaning, castration, vaccination, high temperature andmoving. For such purposes the concentration in the animal feed wouldrange from about 10 to 400 grams per ton, optimally about 200 grams perton.

Further, the compounds of this invention, in aqueous solution orsuspension, may be employed as disinfectants against variousstaphylococci. For this purpose, they are dissolved or suspended inwater, preferably also containing a detergent, at a concentration ofabout 0.5 percent to about 10 percent and may be used as washes todisinfect walls, floors, tables, and the like.

The process of this invention comprises heating a compound of theformulas (FUR; i

wherein R and R are as set forth hereinabove, at a pH of about 6 to l1.5, to a temperature of between about 25 C. and 100 C. Generally thereaction is complete in from 0.5 minutes to 16 hours.

It has been observed empirically, although no explanatory theory can beadvanced as yet, that the process of this invention does not proceedsatisfactorily in a the presence of hydrogen-bonding highly nucleophilicheteroatom functions such as amino, sulfhydryl, phenolic hydroxyl, andthe like. Accordingly, bulfers containing functional groups whichpossess these properties under the reaction conditions employed(temperature, pressure, solvent, and so forth) should preferably beexcluded from the mixture.

Operation in the above-stated pH range may be achieved, for instance, bytreating in the presence of aqueous solutions of hydroxides of alkalimetals, or alkaline earth metals, such as magnesium, calcium, lithium,sodium, potassium, or a buffer solution comprising any of various saltsof carboxylic or mineral acids usually employed for that purpose, as iswell known in the art. For example, such materials as potassiumphosphate, mixtures of boric acid and borax, or of sodium carbonate andsodium bicarbonate may be employed in order to maintain the desired pHof the reaction mixture.

Various solvents may be employed in the process of this invention toassist in the solubilization of the lactones. The use ofdimethylsulfoxide exemplifies a preferred embodiment of the invention.However, any solvent which will solubilize the lactones of Formulas illand IV may be employed. Among such solvents may be mentioneddimethylformamide, ethanol, isopropanol, acetone, acetonitrile,tetrahydrofuran, 1,2- dimethoxyethane, ethylene glycol, and dioxane.Other variations in solvent will readily occur to those skilled in theart.

Generally speaking, the reaction will take place throughout thetemperature and pH ranges specified above. However, optimization ofyield requires that the temperature be increased as the pH is decreased,whereas at a higher pH a lower temperature may be employed. It ispreferred, for instance, when operating in a pH range of about 6 to 8.9,to employ reaction temperatures of about 50 to 100 C. for a time ofabout 0.5 to minutes, and when operating in a pH range of about 8.9 to l1.5, to employ reaction temperatures of about 25 to 50 C. for a time ofabout 0.5 to minutes.

As stated above the compounds of the instant invention may be utilizedas intermediates for the preparation of compounds such as cephalothin,cephalexin and derivatives thereof.

Having obtained a compound of the Formula V S (phenyl)aCNHl 0 v omou thefollowing sequence of reactions may be carried out to form cephalothin.Compound V is reacted with benzoyl chloride either alone or in thepresence of a base (e.g., sodium hydroxide to yield a benzoylatedCompound VI 100K VI In this reaction the trityl group acts as a blockingmoiety and prevents the acylation of the secondary amino group. Thetrityl protecting group is then removed by treating Compound Vl with anaqueous acid, e.g., acetic acid and thereafter N- acylated withZ-thiophene-acetylchloride to yield the compound of Formula VII u NOI-Iz0 C-phenyl COOH VII By reacting this compound with pyridine acetateat a pH of from about 5 to 7, cephaloridine of Formula VIII is obtainedVIII which may be converted to cephalothin by treatment withacetate/acetic acid buffer at a pH of from about 6 to about 8.

Further, Compounds V or Vl may be subjected to hydrogenolysis to yield3-deacetoxy-7-amino cephalosporanic acid, Compound IX COOII IX COOIIdlazomethtme (liphnnyl diszomethuue -CII2OII 1 acetic anhydrlde/pyrldinehydrogenolysis ll+lII O (palladium) X S H2N" O O N CIIO H? CII COOH XIIwherein R is hydrogen or phenyl.

The following examples illustrate the invention EXAMPLE 1 One mg. of3-deacety1-7-l2-(2-thienyl)-acetamido]- cephalosporanic acid lactone isdissolved in 0.2 ml. dimethylsulfoxide and added to 0.8 ml. 0.25 Mpotassium phosphate buffer, pH 8.0. The reaction mixture is placed in awater bath at 100 C. for minutes and then cooled to room temperature inan ice bath. The sample is spotted at 20 .4.1. on Whatman No. 1 paperand chromatographed using ethyl acetatezn-butanol:H,0 (2:5:1) as thedeveloping solvent. The chromatogram is air dried and bioautographedagainst Staphylococcus aureus 209P overnight. A zone of growthinhibition corresponding to that of authentic3-deacetyl-7-[2-(2-thieny1)- acetamido]-cephalosporanic acid is obtained(Rf=0.03). There is no residual 3-deacetyl- 7-[2-(2-thienyl)-acetamido]- cephalosporanic acid lactone. In an unheatedsample (25 C. identically constituted, only 3-decetyl-7-[2(2-thienyl)-acetamido]-cephalosporanic acid lactone is noted (Rf=0.86). An estimateof the yield of 3-deacetyl-7-[2-(2-thienyl)- acetamido]-cephalosporanicacid based on a calibration curve of authentic material gives a resultof percent.

EXAMPLE 2 The procedure of example I is followed except 0.25 M potassiumphosphate buffer, pH 7.5, is used in place of 0.25 M potassium phosphatebuffer, pH 8.0. A yield estimate as in example 1 gives a result of 10-20percent of 3-deacetyl-7-[2-(2- thienyl)-acetamido]-cephalosporanic acid.

EXAMPLE 3 The procedure of example 1 is followed, except 0.25 Mpotassium phosphate buffer, pH 7.0, is used in place of 0.25 M potassiumphosphate buffer, pH 8.0 to obtain 3-deacetyl-7-[2-(Z-thienyl)-acetamidol-cephalosporanic acid. In this example, someresidual 3-deacetyl-7-[2-( 2-thienyl)-acetamido]- cephalosporanic acidlactone is noted.

EXAMPLE 4 The procedure of example 1 is followed except 0.25 M potassiumphosphate, pH 6.5, is used in place of 0.25 M potassium phosphate, pH8.0 to obtain 3-deacetyl-7-[2-thienyl)- acetamido]-cephalosporanic acid.In this example, some residual 3-deacetyl-7-l 2-( Z-thienyl)-acetamido]- cephalosporanic acid lactone is noted.

EXAMPLE 5 The procedure of example 1 is followed except 0.25 M potassiumphosphate, pH 6.0, is used in place of 0.25 M potassium phosphate, pH8.0, to obtain 3-deacetyl-7-[2-(2-thienyl)-acetamidoj-cephalosporanicacid. In this example, some residual3-deacetyl-7-[2-(2-thienyl)-acetamido]- cephalosporanic acid lactone isnoted.

EXAMPLE 6 The procedure of example 2 is followed except that atemperature of 50 C. instead of 100 C. is employed to obtain 3-deacetyl-7-[2-(2-thienyl)-acetamido]-cephalosporanic acid.

In this example, some residual 3-deacetyl-7-[2-(2-thienyl)-acetamidoJ-cephalosporanic acid lactone is noted.

EXAMPLE 7 The procedure of example 2 is followed except that atemperature of C. instead of C. is employed to obtain 3-deacetyl-7-[2-(2-thienyl)-acetamido]-cephalosporanic acid. In thisexample, some residual '-3-deacetyl-7-[2-( 2-thienyl)-acetamidol-cephalosporanic acid lactone is noted.

EXAMPLE 8 The procedure of example 1 is followed except 0.25 M boricacid-borax buffer, pH 8.9, is used instead of 0.25 M potassium phosphatebuffer, pH 8.0, and heating is for 2 minutes to obtain3-deacetyl-7-[2-(2-thienyl)-acetamido]- cephalosporanic acid. Anunheated sample (25 C.) also shows 3-deacetyl-7-[ 2-( Z-thienyl)-acetamido cephalosporanic acid with some residual 3-deacetyl-7-[2-(2-thienyl)-acetamido]-cephalosporanic acid lactone after a 10 minuteincubation period.

EXAMPLE 9 The procedure of example 1 is followed except 0.25 M boricacid-borax buffer, pH,8.0, is used instead of 0.25 M potassiumphosphate, pH 8.0, and heating is for 2 minutes to obtain3-deacetyl-7-[2-( 2-thieny1)-acetamido]- cephalosporanic acid.

EXAMPLE 10 The procedure of example 1 is followed except 0.25 M boricacid-borax buffer, pH 7.0, is used instead of 0.25 M potassium phosphatebuffer, pH 8.0, and heating is for 2 minutes to obtain3-deacetyl-7-[2-(2-thienyl)-acetamido] cephalosporanic acid. In thisexample some residual 3-deacetyl-7-[2-(2-thienyl)-acetamido]-cephalosporanic acid lactone isnoted.

EXAMPLE 1 l EXAMPLE 12 The procedure of example 1 is followed except0.25 M sodium bicarbonate-sodium carbonate buffer, pH 10.0, is usedinstead of 0.25 M potassium phosphate buffer, pH 8.0, and the sample isunheated (25 C.) with a 10-minute incubation period to obtain3-deacetyl-7-[2-(2-thienyl)-acetamido]- cephalosporanic acid.

EXAMPLE 13 The procedure of example 12 is followed except 0.25 M sodiumbicarbonate-sodium carbonate buffer, pH 10.8, is used instead of 0.25 Msodium bicarbonate-sodium carbonate buffer, pH 10.0, to obtain3-deacetyl-7-[2-(2-thienyl)- acetamidol-cephalosporanic acid.

EXAMPLE 14 The procedure of example 1 is followed except dilutepotassium hydroxide (pH 1 1.5) is used instead of 0.25 M potassiumphosphate buffer, pH 8.0, and the sample is unheated (25 C.) with a2-minute incubation period to obtain 3-deacetyl-7-[2-(2-thieny1)-acetamidol-cephalosporanic acid. A yield estimate, as inexample I, gives a result of 10 percent.

EXAMPLE 15 One mg. of 3-deacetyl-7-[2-(2-thienyl)-acetamido}-cephalosporanic acid lactone is dissolved in 0.2 ml. dimethylsulfoxideand added to 0.8 ml. 0.25 M potassium phosphate buffer, pH 8.0. A ,ul.aliquot is spotted on Whatman No. 1 paper and the remaining reactionmixture is immersed in a water bath at 100 C. Ten ul. aliquots areremoved and spotted on the sheet of Whatman No. 1 paper at 1/2, 1, 2, 5,l0 and 30 minutes. The chromatogram is developed with ethylacetate:n-butanol:H,0 (2:5:1) and after air-drying is bioautographedagainst Staphylococcus aureus 209? overnight. The growth inhibitory zonedue to 3-deacetyl-7-[2-(2-thienyl)- acetamide1-cephalosporanic acid isseen in all heated aliquots (Rf- 003) with the maximum zone in the1-minute sample.

EXAMPLE 16 The procedure of example is followed except 0.25 M potassiumphosphate buffer, pH 7.5, is used instead of 0.25 M potassium phosphatebufier. pH 8.0. The maximum zone of growth inhibition is in the 2-minutealiquot.

EXAMPLE 17 The procedure of example 15 is followed except 0.25 Mpotassium phosphate buffer, pH 7.0, is used instead of 0.25 M potassiumphosphate buffer, pH 8.0. The maximum zone of growth inhibition is inthe 5-minute aliquot.

EXAMPLE 18 EXAMPLE 19 Following the procedure of example 1, butsubstituting 3- deacetyl-7-(benzamido)-cephalosporanic acid lactone,there is obtained 3-deacetyl-7-( benzamido)-cephalosporanic acid.

EXAMPLE 20 Following the procedure of example 1, but substituting 3-deacetyl-7-[ 2-( l-naphthyl)-acetamidol-cephalosporanic acid lactone,there is obtained 3-deacetyl-7-[2-( l-naphthyl)-acetamidol-cephalosporanic acid.

EXAMPLE 21 Following the procedure of example 1, but substituting 3-deacetyl-7-[ 2-( 2-pyridyl)-acetamido]-cepl1alosporanic acid lactone,there is obtained 3-deacetyl-7-[2-( 2-pyridyl)-acetamido]-cephalosporanic acid.

EXAMPLE 22 Following the procedure of example 1, but substituting 3-deacetyl-7-[2-(2-furyl)-acetamidol-cephalosporanic acid lactone, thereis obtained 3-deacetyl-7-[2-(2-furyl)-acetamido]- cephalosporanic acid.

EXAMPLE 23 Following the procedure of example 1, but substituting 3-deacetyl-7-(Z-aminoacetamido)-cephalosporanic acid lactone, there isobtained 3-deacetyl-7-(2-aminoacetan1ido)- cephalosporanic acid.

EXAMPLE 24 Following the procedure of example 1, but substituting 3-deacetyl-7-(2-aminopropionamido)-cephalosporanic acid lactone, there isobtained 3-deacetyl-7-(2-aminopropionamido)- cephalosporanic acid.

EXAMPLE 25 Following the procedure of example 1, but substituting 3-deacctyl-7-( 2-amino-2-phenylacetamido )-cephalosporanic acid lactone,there is obtained3-deacetyl-7-(2-amino-2-phenylacetamido)cephalosporanic acid.

EXAMPLE 26 Following the procedure of example 1, but substituting 3-deacetyl-7-[2-(2-pyrryl)-acetamidol-cephalosporanic acid lactone, thereis obtained 3-deacctyl-7[2-( 2-pyrryl)- acetamido]-cephalosporanic acid.

EXAMPLE 27 Following the procedure of example 1, but substituting 3-deacetyl-7-[ 3-( methylsulfinyl )-propionamido]- cephalosporanic acidlactone, there is obtained 3-deacetyl-7-lB-(methylsulfinyl)-propionamidol-cephalosporanic acid.

EXAMPLE 28 Following the procedure of example 1, but substituting 3-deacetyl-7-I 3-( phenylsulfinyl )-propionamido]- cephalosporanic acidlactone, there is obtained 3-deacetyl-7-[3-(phenylsulfinyl)-propionamidol-cephalosporanic acid.

EXAMPLE 29 Following the procedure of example I, but substitutingB-deacetyl-7-[3-benzyl-3-methyl-4-(benzylsulfinyl)-butyramidol-cephalosporanicacid lactone, there is obtained 3-deacetyl-7-[3-benzyl-3-methyl-4-(benzylsulfinyl)-butyramido]-cephalosporanicacid.

EXAMPLE 30 Following the procedure of example 1, but substituting 3-deacetyl-7-benzylaminocephalosporanic acid lactone, there is obtained3-deacetyl-7-benzylaminocephalosporanic acid.

EXAMPLE 31 Following the procedure of example 1, but substituting 3-deacetyl-7-diphenylmethyl aminocephalosporanic acid lactone, there isobtained 3'-deacetyl-7-diphenylmethyl aminocephalosporanic acid.

EXAMPLE 32 Following the procedure of example 1, but substituting 3-deacetyl-7-triphenylmethyl aminocephalosporanic acid lactone, there isobtained 3-deacetyl-7-triphenylmethyl aminocephalosporanic acid.

EXAMPLE 3 3 Conversion of 3-deacetyl-7-[2-(2-thienyl)-acetamido]-cephalosporanic acid lactone to Methyl 7-[2-(2-thienyl)-acetamido]-cephalosporanate To a solution of mg. of3-deacetyl-7-[2-(2-thienyl)- acetamido1-cephalosporanic acid lactone in30 ml. of dimethylsulfoxide is added 120 ml. of ice-chilled 0.01 N KOH(see example 18). After allowing the mixture to stand at roomtemperature for exactly 2 minutes, 7.5 ml. of 0.2 M acetic acid is addedto stop the reaction. Paper chromatography (see example l) of theresulting solution shows the presence of 3- deacetyl-7-[ 2-( Z-thienyl)-acetamido ]-cephalosporanic acid by virtue of its UV-absorption aswell as its antibacterial activity.

The solution which has a pH of 5.0 is extracted three times with BOO-ml.portions of chloroform to remove the DMSO. To the aqueous phase is added80 g. of (Ni-M 50 and the resulting solution is extracted twice with-ml. portions of a solvent mixture consisting of 5 vol. of ethylacetate, 5 vol. of acetone and 1 vol. of water. Paper chromatography(see example 1) shows that the 3-deacetyl-7-[2-( 2-thienyl)-acetamido]-cephalosporanic acid is now extracted in the solvent phase.The combined solvent phase is evaporated under vacuum to a syrup(residual DMSO). Evaporation is repeated twice at a bath temperature of60 C. after the addition of 25 ml. absolute ethanol each time to removethe remaining water. The volume of the syrup amounts to approximately 9ml.

This is dissolved in 40 ml. of ethyl acetate which has been dried overanhydrous Na,SO.. To this solution is added an excess of diazomethane asits solution in ether. After allowing the mixture to stand at roomtemperature for 2 minutes, the excess diazomethane is removed by theaddition of 0.5 ml. of glacial acetic acid. The resulting solution iswashed twice with 25-ml. portions of water to remove the DMSO. Paperchromatography shows that the ethyl acetate solution new contains methyl3-deacetyl-7-[ 2-( 2-thienyl)-acetamido]- cephalosporanate.

The ethyl acetate solution is evaporated under vacuum to dryness. Theresidue is taken up in 14 ml. of pyridine and 7 ml. of acetic anhydrideis added. After allowing the mixture to stand at room temperature for aperiod of about 15 hours, 5 ml. of methanol and 25 ml. of chloroform areadded in the order given. The resulting solution is partitioned with 50ml. of 3 N l-lCl and is then washed twice with 25-m l. portions ofwater. Thin layer chromatography (see below) shows that the chloroformphase now contains methyl 7-[2-(2-thienyl)- acetamido]-cephalosporanate.

The chloroform solution is concentrated under vacuum to a small volumeand chromatographed on 12 8X8-inch cellulose plates in the followingmanner. The precoated cellulose plates (Analtech) are irrigated with amixture of l volume of propylene glycol and 2.3 volumes of methanol inthe same manner as in the development of an ascending chromatogram. Whenthe solvent from reaches the upper edge of the plates, they areair-dried for 30-60 minutes. The sample is applied as a streakthree-fourths inch from the lower edge of the plates. An ascendingchromatogram is then developed with propylene glycol saturated tolueneas the mobile solvent. The band recognized as the desired methyl7-[2-(2-thienyl)- acetamidol-cephalosporanate by virtue of theUV-absorption and its Rf (0.6) is eluted with a 1:1 methanol-chloroformmixture. The eluate is partitioned between equal volumes of chloroformand 50 percent (by volume) methanol in water. The chloroform phase iswashed twice with equal volumes of 25 percent methanol in water and thendried over anhydrous Na SO On evaporating the chloroform solution todryness under vacuum, 9 mg. of crude crystalline methyl 7-[2-( 2-thienyl)-acetamido]-cephalosporanate is obtained. it is recrystallizedfrom methanol to give the pure product, having an IR spectrum identicalto that of authentic methyl7-[2-(2-thienyl)-acetamido]-cephalosporanate.

EXAMPLE 34 Diphenylmethyl ester of 7-tritylamirio-3-(hydroxymethyl)-ceph-3-em4-carboxylic acid To a solution of 454 mg. of7-tritylamino-3-(hydroxymethyl)-ceph-3-em-4-carboxylic acid lactone in50 ml. acetone at 20*- C. is added 333 ml. of 0.003 N aqueous potassiumhydroxide during 20 (:5) minute interval. After an elapsed time of 30minutes, the reaction mixture is treated with an aqueous solution ofp-toluene-sulfonic acid at 5 C. until the pH of 5.0 is attained. Theresulting mixture is then evaporated at reduced pressure at 5 C. Theresidual hydroxy acid is taken up in ice-cold dimethyl formamide andtreated with approximately 1 ml. of a one molar solution [cf. Aboderin,Delpiene, and Fruton, J. Am. Chem. Soc., 87, 5469 (1965)]ofdiphenyl-diazomethane in dimethylformamide until a slight color ofexcess diazocompound persists. The excess reagent is destroyed by addinga small amount of acetic acid. (The unstable hydroxy ester may beconveniently used as this solution.)

EXAMPLE 35 7-Aminocephalosporanic acid The solution from example 34, at0 C., is treated with l g. of acetic anhydride and 1 ml. pyridine andallowed to warm to room temperature for l5 hours. The reaction mixtureis then poured over ice and the pH of the mixture adjusted to 7.5. Thesystem is extracted with ether to isolate the crude product. Theethereal extract is washed twice with cold 1 percent aqueous sodiumbicarbonate solution, then with saturated aqueous sodium chloridesolution and finally dried and evaporated at l 0 C. to dryness byreduced pressure.

Catalytic hydrogenolysis of the material may be conducted in an ethylacetate (or methanol) solution (ca. 50 ml.) over 1.0 g. of 10 percentpalladium on carbon catalyst at room temperature for l to 1.5 hours. Thehydrogenated mixture is treated with excess ice-cold 1 percent aqueoussodium bicarbonate solution (ca. 50 ml. the aqueous layer is washed oncewith ether, then diluted with 25 ml. glacial acetic acid. After stirringthe mixture for 1 hour at room temperature, the solution is evaporatedat reduced pressure. Trituration of the residue with ether removes thetriphenylcarbinol byproduct and deposits crude 7-aminocephalosporanicacid.

EXAMPLE 35 3-Deacetoxy-7-aminocephalosporanic acid When the catalytichydrogenolysis in example 34 is allowed to proceed in methanol solutionuntil nearly 3 molecular equivalents of hydrogen are absorbed (at l to 4atmospheric pressure), the product, after working up as in example 34,consists mainly of 3-deacetoxy-7-aminocephalosporanic acid.

EXAMPLE 36 Preparation of 3-(benzoyloxymethyl)-7-aminoceph-3-em-4-carboxylic acid To a solution of 454 mg. (l.0 mmole) 7-tritylamino-3-(hydroxymethyl)-ceph-3-em-4-carboxylic acid lactone in 50 ml. acetone at20 C. is added 333 ml. 0.003 N aqueous potassium hydroxide as in example34. Afler 30 minutes, the rapidly stirred solution is treated with 164mg. (1.3 mmole) benzoyl chloride at 0 C. with the slow addition of 0.03N aqueous potassium hydroxide over a 15-minute interval to maintain pl-l:05 After an additional 30 minutes at 0 C., the solution is acidified topH 3 with formic acid and stirred for 25 minutes, after which time thepH is adjusted to 4.6 with dilute aqueous potassium hydroxide. Thedesired product is separated by filtration. Trituration of the residuewith ether leaves the hydrochloride salt of the product.

EXAMPLE 37 Benzyl ester of 7-tritylamino-3-(hydroxymethyl)-ceph-3-em-4-carboxylic acid Substitution of phenyldiazomethane in lieu ofdiphenyldiazomethane in example 34 produces the desired product.

EXAMPLE 38 Benzyl ester of 7-aminocephalosporanic acid A 10 percentsolution of the crude benzyl ester in the preceding example in pyridineis treated with a 10-fold excess of acetic anhydride at roomtemperature. The reaction mixture is evaporated at l0 C. at reducedpressure and the resulting residue stirred with excess aqueous 1 percenthydrochloric acid for one-half hour at ambient temperature. The resultis evaporated under reduced pressure, yielding the desired product.

EXAMPLE 39 Bis-(p-methoxyphenyl)methyl ester of 7-tritylamino 3-hydroxymethylceph-3-em-4-carboxylic acid Following the procedure ofexample 34, but utilizing bis-(pmethoxyphenyhdiazomethane in lieu ofdiphenyldiazomethane, the desired product is recovered.

EXAMPLE 40 7-Aminocephalosporanic acid The product recovered byutilizing the product of example 39 and treating it in accordance withthe procedure of example 35 is treated with 1:1 acetic acid-watersolution for about 30 minutes at ambient temperature with rapidstirring; the solid formed is removed by filtration and triturated withether. The final solid residue is 7-aminocephalosporanic acid.

EXAMPLE 41 Deacetoxy-7-aminocephalosporanic acid To a solution of 454mg. 7-tritylamino-3-(hydroxymethyl)- ceph-3-em-4-carboxylic acid lactonein 50 ml. acetone at 20 C. is added 333 ml. 0.003 N aqueous potassiumhydroxide as in example 34. After about 30 minutes the solution isconcentrated to about 30 ml. at reduced pressure at about 20 C. Theresulting solution is chilled to about 5 C. and treated, with very rapidstirring, with 164 mg. benzoyl chloride for 45 minutes; during thereaction, dilute potassium hydroxide is added as needed to maintain pl-l8.0105. The reaction mixture is then treated with 1 gram Norite(activated carbon) and filtered. One gram of percent palladium on carbonis added and the suspension hydrogenated for 1.5 hours at 3 atmospherespressure. The reaction mixture is readjusted to pH of about 7.5 to 8 ifnecessary and filtered. The pH of the filtrate is adjusted to 4.6 andchilled to precipitate the product. Trituration with several smallportions of water leaves the product as a powder.

What is claimed is:

1. A process for preparing compounds having the formulas:

which comprises treating a compound selected from the group consistingof:

with a solution having a pH of from about 6 to about 11.5, wherein R isa member selected from the group consisting of alkyl having up to sevencarbon atoms, phenyl, X-substituted phenyl, naphthyl, X-substitutednaphthyl, pyrridyl, pyrryl, furyl, thienyl, pyrridyl-lower alkyl,pyrryl-lower alkyl, fury]- lower alkyl, thienyl-lower alkyl,

Ll J.

wherein R is a member selected from the group consisting of alkyl havingup to seven carbon atoms, phenyl X -substituted phenyl, naphthyl,X-substituted naphthyl, pyrridyl, pyrryl, furyl, thienyl, pyrridyl-loweralkyl, pyrryl-lower alkyl, furyllower alkyl, and thienyl-lower alkyl; Ris a member selected from the group consisting of lower alkyl,monocyclic aryl, and monocyclic aryl-lower alkyl; R and R each areselected from the group consisting of hydrogen, lower alkyl, monocyclicaryl, and monocyclic aryl-lower alkyl; n is l, 2, or 3; and X is amember selected from the group consisting of lower alkyl, lower alkoxy,and halo; and R is a member selected from the group consisting oftriphenylmethyl, diphenylmethyl, and benzyl; and recovering the productsof such treatment.

2. A process in accordance with' claim 1 wherein the treatment iscarried out at a temperature of from about ambient to C.

3. A process in accordance with claim 1 wherein the treatment is carriedout in the presence of a solvent.

4. A process in accordance with claim 2 wherein the treatment iseffected in the presence of potassium hydroxide, sodium hydroxide,lithium hydroxide, magnesium hydroxide, or other suitable alkalinecompounds.

5. The process of claim 2 in which the treatment is effected in thepresence of a buffer solution.

6. A process in accordance with claim 1 wherein the conditions oftreatment comprise a pH in the range of about 6 to about 8.9, and areaction temperature of about 50 C. to about 100 C.

7. A process in accordance with claim 1 wherein the conditions oftreatment comprise a pH range of about 8.9 to about 1 1.5 and a reactiontemperature of about 25 C. to about 50 C.

8. A process in accordance with claim 1 wherein the starting material is3-deacetyl-7-[2-(2-thienyl)-acetamido]- cephalosporanic acid lactone'.

9. The process of claim 1 wherein the starting material is 3-deacetyl-7-triphenylmethyl aminocephalosporanic acid lactone.

10. A process for preparing 3-deacetoxy-7- aminocephalosporanic acidwhich comprises treating a compound of the formula:

wherein R is defined as in claim 1 with a solution having a pH of about6 to l 1.5 to form an acid having the formula:

wherein R is as defined in claim 1 and R is hydrogen or phenyl,acylating the hydroxyl group of said compound and hydrogenolyzing theproduct thus formed to yield 7-acylamido cephalosporanic acid.

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,1"11 the Abstract--that portion 9f the 2nd formula reading should read:I O :LN I O I 0.

Signed and sealed this 23rd day of May 1912a {SEJ-LLI? fittest:

EDWARD MFLETCHER JR ROBERT GOTT SGHALK Attes'oing Qfficer Commissioner01 Patents

2. A process in accordance with claim 1 wherein the treatment is carriedout at a temperature of from about ambient to 100* C.
 3. A process inaccordance with claim 1 wherein the treatment is carried out in thepresence of a solvent.
 4. A process in accordance with claim 2 whereinthe treatment is effected in the presence of potassium hydroxide, sodiumhydroxide, lithium hydroxide, magnesium hydroxide, or other suitablealkaline compounds.
 5. The process of claim 2 in which the treatment iseffected in the presence of a buffer solution.
 6. A process inaccordance with claim 1 wherein the conditions of treatment comprise apH in the range of about 6 to about 8.9, and a reaction temperature ofabout 50* C. to about 100* C.
 7. A process in accordance with claim 1wherein the conditions of treatment comprise a pH range of about 8.9 toabout 11.5 and a reaction temperature of about 25* C. to about 50* C. 8.A process in accordance with claim 1 wherein the starting material is3-deacetyl-7-(2-(2-thienyl)-acetamido)-cephalosporanic acid lactone. 9.The process of claim 1 wherein the starting material is3-deacetyl-7-triphenylmethyl aminocephalosporanic acid lactone.
 10. Aprocess for preparing 3-deacetoxy-7-aminocephalosporanic acid whichcomprises treating a compound of the formula: wherein R1 is defined asin claim 1 with a solution having a pH of about 6 to 11.5 to form anacid having the formula: wherein R1 is as defined in claim 1, reactingthis acid with benzoyl chloride, recovering a compound wherein R1 isbenzoyl, and hydrogenating the benzoyl compound and recovering3-deacetoxy-7-amino cephalosporanic acid.
 11. A process for preparing a7-acylamido-cephalosporanic acid which comprises reacting the acidcompound of claim 10 with a compound selected from the group consistingof diazoMethane and diphenyl diazomethane, recovering a compound of theformula: wherein R1 is as defined in claim 1 and R6 is hydrogen orphenyl, acylating the hydroxyl group of said compound andhydrogenolyzing the product thus formed to yield 7-acylamidocephalosporanic acid.